The main focus of this project is to define the potential autocrine/paracrine mechanisms regulating the growth of human tumors and utilize these as rational targets for early detection and intervention of malignant disease. We have developed several investigative strategies to achieve this goal. Our initial effort was accomplished by acclimating tumor cell lines to grow in protein-free/peptide-free medium (R0), thereby forcing the cell to express maximum survival capabilities in a nutrient poor environment. RPMI-1640 without phenol red was used as the base medium supplemented with L-glutamine (2nM) and sodium selenite (30nM). Under these conditions we have been able to establish long-term growth (2-5 yrs) from over 50 epithelial cancer lines including those of: lung (small cell/non-small cell), breast, colon, ovarian, pancreatic, prostate, and neuroblastoma/glioblastoma lineage. During the adaptation process all cells express a varying latency or acclimation period of between 5-14 days before abundant growth is achieved. This delay is thought to reflect the time required for gene activation and the accumulation of endogenous growth factors to critical levels since R0 conditioned media supports the growth of unadapted cells without a proliferative lag phase. Biochemical analysis of the conditioned media from R0 cells revealed the presence of protein/peptides such gastrin releasing peptide (GRP), insulin-like growth factors (IGF-I/IGF-II), transforming growth factor-alpha (TGF-a), adrenomedullin (AM) and transferrin (Tf) which are established mitogens of tumor. Since R0 base medium contains no exogenous protein/peptides, the only source of such products was for the R0 adapted cells themselves. These conditions may mirror steps in carcinogenesis which involve clonal expansion of the initiated cell. For proliferation to occur in any cell process (carcinogenesis, wound repair, or embryogenesis) two fundamental requirements are necessary: 1) Activation of a signal transduction pathway (i.e. cAMP, Ca+2 flux, PI turnover, phosphorylation) by an inducer factor (IF) which triggers initial cell growth mechanisms and 2) Intracellular cofactor (metal ions and vitamins) availability mediated by a competency factor (CF) which allows growth to take place. It is the interplay between these two factors (IF/CF) that drives proliferation and underlies the clonal expansion process. Thus by identifying the IF/CF and their respective receptor systems expressed during R0 adaptation of tumor cell lines we have been able to generate appropriate molecular and immunological reagents to evaluate their involvement during normal to malignant conversion in pathological specimens. In addition, since disruption of IF/CF interactions result in growth cessation, these factors make potential targets to intervene in the carcinogenesis pathway by blocking the promotion event. Interestingly, AM has now been proven to be a direct mitogen of epithelial cancer cells, function as an inducer of neovascularization and mediate the inhibition of programmed cell death, representing features which encompass the definition of a tumor promotion factor. Recent studies using our R0 approach have identified an AM binding protein produced by human tumor cell lines adapted to R0 conditions. Followup studies have demonstrated this AM binding protein to be complement facator H (fH) which is a serum protein capable of disrupting the complement cascade and protecting cells from lysis. Hence, R0 adapted cancer cells maybe producing a protective factor (fH) which could ultimately allow tumors to circumvent immune surveillance mechanisms.